Color developing composition consisting of an enzyme,a salicylate and a hypochlorite donor



Nov. 24, 1970 I s cY 3,542,649

COLOR DEVELOPING COMPOSITION 'CONSISTING OF AN, ENZYME, A SALICYLATE ANDA HYPOCHLORI'IE DONOR Filed. April 7; 1967 2 Sheets-Sheet 1 SAMPLE o a IPLATE 0 o TUBING GLASS COIL ML/MIN 91A RECORDER \5 MM F/c FLOWCELLCOLORIMETER FIG. I

Nov. 24, 1970 R. L. SEARCY 3,542,649

COLOR DEVELOPING COMPOSITION CONSISTING OF AN ENZYME, A

SALICYLATE AND A HYPOCHLORITE DONOR Filed April .7, 1967 2 Sheets-Sheet.2

0 'I I I I I I I 0 20 40 so 80 I00 I20 I40 I60 UREA NITROGENCONCENTRATION (mg./lO0mI.)

FIG. 3

United States Patent Ofice Patented Nov. 24, 1970 US. Cl. 195-1035 3Claims ABSTRACT OF THE DISCLOSURE The present invention relates to anautomated system for the an alytical determination of urea nitrogen inblood serum, plasma or in urine and to a color developing compositionuseful in the system consisting of an enzyme, a salicylate, preferablyan alkali metal salicylate, and a hypochlorite donor, preferably sodiumor potassium dichloroisocyanurate.

BACKGROUND OF THE INVENTION The determination of urea in blood serum,plasma or urine has been accomplished by several techniques. In one suchprior art technique, serum, plasma or diluted urine is incubated withbuffered urease for conversion of urea to ammonia. The ammonia is thenmeasured by colorimetry which is based upon the reaction of the ammoniaand reagents such as sodium phenate and hydrochlorite to form a coloredcomplex. The intensity of the color developed is proportional to theammonia concentration. This procedure has been proposed for use inautomated analyzers.

Other methods which are routinely employed for the determination of ureain biological fluids are also available. One of the more commonly usedmethods involves hydrolizing urea to ammonia carbonate by means of theenzyme urease in the presence of a buffered solution. Ammonia isliberated from the carbonate salt by the addition of sodium borate andthen distilled into 0.05 N hydrochloric acid. The amount of nitrogenpresent is then determined colorimetrically after nesslerization. Thisprocedure is described in detail in the Manual of Clinical LaboratoryMethods, Fourth Edition by Opal Hepler, Publisher, Charles C. Thomas,Springfield, Ill.

Another commonly employed method relies upon the amount of yellowpigment formed by condensing diacetylmonoxime with urea in acid solutionin a filtrate of the fluid which is protein free. This procedureiscurrently utilized in automated analytical techniques. A description ofdiacetylmonoxime methodology appears in Marsh et a1, Amer. J. Clin. Path28, 681, 1957.

More recently, it has been found that the determination of urea inbiological fluids can be manually accomplished utilizing a colordeveloping composition comprising sodium salicylate, sodiumnitroprusside and sodium dichloroisocyanurate.

All of the prior art techniques suflered from certain defects. Forexample, the last-mentioned method while suitable for manual proceduresdid not develop color of suitable intensity to admit of its use inautomated analyzers. Additionally, sodium nitroprusside is sensitive toatmosphere and could conceivably form toxic HCN. Other of the techniquesare concentration dependent and require a large sample size.Furthermore, time is also a critical factor in the use of some of suchprior art procedures and hence, extreme caution is required by the userif results are to be meaningful. Also, certain of the reagents utilizedin prior art techniques were found to be unstable, further limiting theusefulness of the procedure which employs same.

It is an object of this invention to provide an automated system fordetermining urea nitrogen in biological fluids and materials usable inthe system which do not suffer from any such defects.

BRIEF SUMMARY The present invention relates to a reagent combination forthe automatic analysis of urea nitrogen in a specimen of a biologicalfluid which consists essentially of mixing the specimen with an amountof enzyme which reacts with the urea in the biological fluid to liberateammonia, dialyzing the so-formed solution against a salt of a salicylateand developing color by adding a hypichlorite donating agent, to therecipient stream of the dialysis.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a schematic flow diagramillustrating a novel automated system for automatically analyzing ureanitrogen in biological fluids utilizing the novel color developingcombination of the prsent invention.

FIG. 2 is a recording of the photometric response obtained whenutilizing the automated system of FIG. 1.

FIG. 3 is a plot in terms of optical density of the photometricresponses illustrated in FIG. 2.

DETAILED DESCRIPTION The present invention in detail relates to aprocedure for the chemical determination of urea nitrogen in bio logicalfluids such as blood serum, plasma or urine.

In FIG. 1, an automated testing system is shown schematically wherein aspecimen sample to be tested which may be oxalated blood or urine isdrawn up in sequence from separate depressions in the sample plate whichrotates at a constant speed to provide the system with 60 specimensamples per hour. A sample, so drawn, is mixed with an enzyme,preferably urease, which reacts with urea in biological fluids toliberate ammonia. The mixture is passed through a glass incubation coilof conventional design maintained at 37 C. After the mixture is passedthrough the glass incubation coil, it is next pumped into a dialyzermodual that is provided at its entrance with a small cellophane membraneor the like for dialysis on the opposite side of the membrane againstthe salicylate color reagent, preferably, an alkali metal salicylate,most advantageously, sodium salicylate. The residual, non-diffusableportion of the sample-urease mixture is discarded. The hypochloritedonating reagent, (preferably, an alkaline solution of an alkali salt ofdichloroisocyanurate, most preferably sodium dichloroisocyanurate) isadded to the recipent stream of dialysate. After mixing the resultingcbmbination in a suitable mixer, color developed at 37 during transitthrough an incubation coil. Photometric measurements are then performedat 660 mg. in a 15 mm. floWcell colorimeter, i.e. the absorbance of thesolution to be tested is measured at 660 millimicrons in aKlett-Summerson colorimeter using a red filter (No. 660) against areagent blank. The results obtained are recorded on a conventionalrecording mechanism.

The system illustrated in FIG. 1 aspirates at a rate of 60specimens/hour. The rate of flow in ml./min. of the materials enteringthe system according to a preferred technique is illustrated in FIG. 1.The materials entering the system are pumped into it by any suitablepumping means adjusted to maintain the rate of flow illustrated inFIG. 1. The mechanism for the system of the present invention can beconveniently provided by a modification in accordance with the systemillustrated in FIG. 1 of the manifold of the Technicon Auto Analyzerwhich uses the diacetylmonoxime procedure for determining urea nitrogenin biological fluids and the system disclosed in US. Pat. No. 2,879,141issued Mar. 24, 1959.

FIG. 2 is a representative recording of the results obtained with theautomated system depicted in FIG. 1 utilizing sample solutionscontaining urea nitrogen ranging in concentrations of from 5 to 150 mg.per 100 ml.

When these results are plotted in terms of optical density, they exhibita linear relationship to urea nitrogen levels at the concentrationsexamined as shown in FIG. 3.

In an alternate procedure, aliquots of ammonia-free distilled water areplaced between each specimen on the sample plate illustrated in FIG. 1to preclude cross contamination.

Any enzyme which is capable of reacting with urea to liberate ammonia issuitable for the purposes of the present invention. Most preferred,however, is urease. Urease preparations suitablt'for the purposes of thepresent invention are readily available commercially in states ofgreater or lesser purity. The preferred urease preparations to beemployed with the automated technique which constitutes the presitntinvention (as illustrated in FIG. 1) have an activity of from about 0.8to about 1.0 Sumner units per mg. as measured at 30 C. When employed inthe method of the invention, the enzyme urease is put in solution. Thesolution is buffered to a pH which favors enzyme activity and stabilityutilizing conventional buffers such as monopotassium dihydrogenphosphate, disodium monohydrogen phosphate and ethylenediaminetetraacetic acid and salts thereof. Particularly advantageous resultsare realized when a chelating agent such as an alkali metal salt ofethylene diamine tetraacetic acid, preferably, disodium ethylene diaminetetraacetate, is employed as the buffer.

A particular preferred buffered urease solution suitable for thepurposes of the present invention is obtained by dissolving grams ofdisodium ethylene diaminetetraacetate and 200 mg. of urease (activityequal to 0.8 modified Sumner units per mg.) in about 750 ml. ofammoniafree distilled water. The resulting mixture is adjusted to pH 6.5with a small amount of 2.5 N sodium hydroxide and the volume brought to1 liter with additional deionized water. The enzyme solution is readyfor use in the automated system illustrated in FIG. 1. The so-preparedenzyme solution, if desired, may be stored indefi nitely at C.

A preferred salicylate color reagent utilized in the mechanism depictedschematically in FIG. 1 is prepared by dissolving 170 grams of sodiumsalicylate in 1 liter of ammonia-free distilled water. This reagent canbe used immediately in the system illustrated in FIG. 1. Alternatively,it can be stored for later use. It is found stable for many months whenstored in glass at room temperature.

While sodium salicylate is indicated above as being the mostadvantageous salicylate salt for the purpose of the present invention,the use of other salicylate salts capable of effecting the desired endwith equal efiicacy are contemplated. Thus, for the purposes of thepresent invention, there can be utilized 4-amino salicylate, phenylsalicylate, calcium salicylate, zinc salicylate, strontium salicylate,copper salicylate, lithium salicylate, magnesium salicylate, ammoniumsalicylate and salicylaldoxime. With the exception of phenyl salicylateand salicylaldoxime, a solution containing any of the above, can beprepared by the same procedure described above in connection withpreparation of the most advantageous salicylate color reagent containingsodium salicylate. However, since phenyl salicylate and salicylaldoximeare not particularly soluble in water, when such are utilized, they aredissolved in 1 liter of 1.0 N sodium hydroxide, rather than 1 liter ofammonia-free distilled water as described above in connection with thepreparation of the sodium salicylate color reagent.

The preferred hypochlorite donor utilized in the system schematicallyillustrated in FIG. 1 is prepared by dissolving 5.0 gm. of sodiumdichloroisocyanurate in 1 liter of 0.6 N sodium hydroxide. Theso-prepared alkaline donor medium can be used immediately oralternatively, it can be stored for use at some subsequent time. Thedonor medium resists deterioration when stored in glass at refrigeratortemperature, i.e., 5 C.

Representative of other salts of dichloro isocyanurates equallyefficacious for the purposes of the present invention is the potassiumsalt of dichloroisocyanurate.

In alkaline solutions, the salt of dichloroisocyanurate dissociates toproduce hypochlorite ions and, is therefore, functioning as thehypochlorite donor. The developed color is due to the propensity ofammonia to react with the hypochlorite ions to form an intermediatewhich, when a salicylate is present, forms a chromatic substance. Thechromatic substance obtained when utilizing sodium salicylate in thesystem illustrated schematically in FIG. 1 takes a brilliant emeraldgreen color.

Utilizing other salicylates, chromogenic responses obtained are asfollows:

REACTIVITY OF VARIOUS SALICYLATES WITH AMMONIA Compound: Chromogenicresponse:

Amino salicylate green Phenyl salicylate blue Calcium salicylate greenZinc salicylate green Strontium salicylate green Copper salicylate greenLithium salicylate green Magnesium salicylate blue green Ammoniumsalicylate green salicylaldoxime brownish green In testing for thepresence of urea nitrogen, the invention described herein will beemployed in the following manner. First, the enzyme, e.g. the bufferedurease solution prepared as described above and the sample are pumpedinto the system illustrated schematically in FIG. 1 whereby a mixture ofthe two is obtained. The resultant mixture is then incubated, afterwhich it flows into the salicylate color reagent entering the system.The so-prepared combination then flows along the system to a pointwhereat the hypochlorite donating agent meets therewith. An immediatecolor formation occurs. The optical density developed by the so-formedmixture is measured by the use of an accurate measuring instrument suchas spectrophotometer or colorimeter which measures the transmission oflight or other suitable radiation through the resultant medium. By theapplication of Beers law, the concentration of the color compoundpresent can be determined. From a comparison of this concentration to astandard solution, the original concentration of urea present iscalculated and recorded. A particular salient feature of the use of thesalicylate and hypochlorite donating agent exclusively as describedabove is the rapidity in which the color formation occurs. Thus, by thenovel color forming composition of the present invention, a technique isprovided which is eminently well-suited for an automated system and yetavoids the problems inherent in prior art automated systems designed toefifect this end.

The reaction with urea nitrogen according to the present invention ishighly sensitive, specific and reproducible.

What is invented is a particularly efiicacious method for measuring ureanitrogen automatically in biological fluids by the simple techniquedisclosed. In its use, there is avoided materials which may be causticto equipment and hence, cause corrosion problems. Furthermore, the useof nitroprusside is avoided. Under certain conditions, this substancecan form HCN, a gas which is highly toxic and dangerous. Thus, the useof a substance which could conceivably effect the health and well-beingof the technicians utilizing the mechanism illustrated in FIG. 1 isavoided. Furthermore, if nitroprusside were to be utilized in theautomated technique schematically illustrated in FIG. 1, the system isno longer suited for testing rapidly many samples of specimen. Thus, theauto mated urea nitrogen technique utilizing only a salicylate and ahypochlorite donating reagent has been unexpectedly found to utilizereagents that are neither hazardous or dangerous to equipment.Additionally, the discard resulting from the operation of the systemillustrated schematically above is nonacidic in nature. Hence, wastematerials may be easily disposed of.

Several distinct advantages over available automated methods for ureanitrogen analysis are provided by the new technique. The diacetylmonoxime procedure, for example, entails the use of a hot mixture ofconcentrated sulfuric and phosphoric acids. This solution is not onlyhazardous to personnel, but it is also highly corrosive and presents aserious disposal problem. Furthermore, the results obtained with thistechnique are not linear and hence, Beers law is not satisfied.

Also, there have been advocated for automated urea nitrogen assay theuse of phenate-hypochlorite reagent. This mixture adversely afiects thepolyvinyl chloride (Tygon) transmission tubing conventionally used insuch system as well as the methyl methacrylate (Lucite) dialyzer platesalso conventionally utilized in the system. Therefore, glass tubing mustbe used from the point of entry of phenatehypochlorite reagent into therecipient stream to the position where the mixture leaves thecolorimeter.

To summarize briefly, the present invention relates to a diagnostic testfor the detection of urea nitrogen in biological solutions which ishighly accurate and entails the use of reagents that are neitherhazardous nor damaging to instrumentation.

I claim:

1. A method for the automatic analysis of urea nitrogen in a specimen ofa biological fluid which consisting essentially of adding the specimento an amount of urease which reacts with urea nitrogen in saidbiological fluid to liberate ammonia, dialyzing the so-formed solutionagainst a salt of a salicylate and developing color by adding ahypochlorite donating agent to the recipient stream of the dialysate.

2. A method for the automatic analysis of the urea nitrogen in aspecimen of a biological fluid as defined in claim 1 wherein the salt ofa salicylate utilized is an alkali metal salt thereof and thehypochlorite donating agent utilized is an alkali metal salt ofdichloroisocyanurate in an alkaline medium.

3. A method for the automatic analysis of urea nitrogen in a specimen ofa biological fluid as defined in claim 1 wherein the salt of asalicylate utilized is sodium salicylate and the hypochlorite donatingagent utilized is sodium dichloroisocyanurate in an alkaline medium.

References Cited UNITED STATES PATENTS 2,797,149 6/1957 Skeggs..195--103.5 X 2,879,141 3/1959 Skeggs 23-253 3,432,395 3/1969 Reardon103.5

ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 195-127

